The invention relates to a method for injecting a liquid sample in an HPLC analyzing device. The invention also relates to an apparatus for injecting a liquid sample in an HPLC analyzing device. The invention furthermore relates to a valve assembly for use in such a method and in such an apparatus.
Such a method is commonly known and used in High Pressure Liquid Chromatography (HPLC), also called High Performance Liquid Chromatography. In normal HPLC, the pressure may be as high as up to around 200 bar. In case of higher pressures, up to around 1000 bar or even higher, HPLC is often more specifically referred to as Ultra High Pressure (or Performance) Liquid Chromatography (UHPLC). The invention generally relates to HPLC, and more particularly to UHPLC.
Upon completion of the said step of initializing the apparatus, the valve assembly is in the second valve position, which means that the sample loop is part of the second liquid circuit then. The second circuit, including the sample loop thereof, is containing the mobile phase then. The initial quantity of the sample has been supplied to the first liquid circuit for example by means of aspirating the sample via a needle out of a sample holder. Often, the sample holder has different compartments containing different samples. The first circuit typically is also connected to a washing liquid container and to a waste container. The liquid pressure in the first circuit is relatively low, usually equalling environmental pressure, thus giving rise to a pressure difference with the pressure of the mobile phase in the second circuit, from which second circuit there has to be injected, at a later stage, liquid with high pressure into the analyzing device.
After completion of said initializing step, the valve assembly is switched from the second valve position into the first valve position. During this switching of the valve assembly, the sample loop is losing its role as being a part of the second circuit and is attaining a role as being part of the first circuit.
In the newly attained first valve position, an additional quantity of the sample is supplied into the first circuit to such an extent that the sample loop is at least partly filled with the sample. This may for example be achieved by means of aspirating, via the needle, additional sample out of the sample holder.
By consecutively switching the valve assembly back into the second valve position, the sample in the sample loop is contained in the second circuit, so that the sample together with the mobile phase can be injected from the second circuit into the analyzing device.
A disadvantage encountered in such known methods, is explained as follows. During the abovementioned switching, directly after completion of said initializing step, of the valve assembly from the second valve position into the first valve position the high pressure mobile phase contained in the sample loop is first being cut-off from high pressure mobile phase contained in remaining sections of the second circuit. Next during the said switching, said cut-off high pressure mobile phase is being inserted into the first circuit which contains low pressure liquid. The result is that the high pressure mobile phase upon its insertion will expand in the first circuit and will therefore pass at least the first valve inlet, thus driving away the sample in the direction of the sample supply means. Thus, an uncontrolled quantity of the sample will flow out of the first circuit via the sample supply means (for example a needle).
This uncontrolled outflow is undesirable for at least the following reasons. At first, it may lead to contamination and/or cross-contamination of a sample holder. Furthermore, due to the uncontrolled outflow, the quantity of the sample between the sample supply means and the first valve inlet is not accurately known anymore. This results in problems with dosing the sample, especially in cases where the sample loop has to be only partially filled with the sample by the said supplying of the said additional quantity of the sample into the first circuit.